PHYCOBILIN COVALENT COUPLED TO β SUBUNIT CYS-155 OF PHYCOCYANIN AND PHYCOERYTHROCYANIN FROM MASTIGOCLADUS LAMINOSUS PCC 7603

Phycobiliproteins, as a homologous family of light-harvesting proteins present in cyanobacteria, red algae and cryptophytes, are the function composition of light-harvesting complexes in the algae photosynthesis. The phycobiliprotein biosynthesis is the phycobilin addition to the apophycobiliproteins. So far, the correct attachments of most chromophores are catalyzed by lyases, of which only few have been characterized in vivo. In Mastigocladus laminosus PCC7603, phycocyanin and phycoerythrocyanin have two subunits, the coupling sites of phycobilin addition to the apophycobiliprotein of β subunits are Cys-84 and Cys-155. The protein cpcS1 encoded by gene alr0617 was proved to be the lyase of Cys-84. To search the lyase of Cys-155, four genes were filtrated by BLAST comparison. They were cpcT1, cpcT2, cpcS1 and cpcS2, in which genes cpcT1 and cpcS2 had to be transformed to the vector pCDFDuet for the experiments need by molecular cloning technique, and the correct clones could be selected by agarose gel electrophoresis and SDS-PAGE. The entire pathway of a fluorescent β subunit was synthesized from combinational expression of four genes in three compatible pACYCDuet-ho1-pcyA, pET30a-cpcB (C84S) or pET30a-pecB (C84A) and one of the filtrated four genes in E. coli, in LB culture medium at 20 ℃ for 12h. After mechanical disruption, the reconstitution protein were purified by metal chelating affinity chromatography, and dialysed in buffer overnight to leach the medical irons. At last, they were confirmed by lyase activity comparisons, SDS-PAGE, chromoprotein denaturation, absorbance and fluorescence spectrum. As a result, compared to the related literatures of PCB spectrum, the protein cpcT1 encoded by gene all5339 was the lyase of Cys-155. At the same time, it proved that the other three genes have no effects to the Cys-155. Up to now, the two lyases which could catalyze the coupled reaction of the apophycobiliprotein’s two sites of β subunits and PCB were all found. This result will be signality for researching the phycobiliprotein biosynthesis, light-harvesting principle in photosynthesis, assembling phycobilisome and so on.